Role of the ubiquitin-like modifier FAT10 in protein degradation and ...

More documents

Recommendations

Info

Chapter 3 Figure 20: Both full length HDAC6 as well as the isolated CAT1 and BUZ domains interact with FAT10 in vitro. The diglycine motif of FAT10 is not required for its interaction with HDAC6. (A) GST-pulldown of [ 35 S]-methionine labeled in vitro transcibed and translated HDAC6 mutants with recombinant FAT10. Bound proteins were analyzed by SDS-PAGE and autoradiography. CAT1 = first catalytic domain, BUZ = ubiquitin binding zinc finger. (B) Coomassie-staining of the recombinant proteins used in A. (C) GST-pulldown of [ 35 S]-methionine labeled HDAC6 with recombinant wild-type FAT10 or FAT10 lacking the C-terminal diglycine motif (∆GG). (D) Coomassie-staining of recombinant FAT10 used in C. The catalytic activity of HDAC6 is not required for interaction with FAT10 The role of the deacetylase activity of HDAC6 in aggresome formation remains somewhat mysterious. On one hand, reintroduction of either the ∆BUZ or a catalytically inactive mutant into HDAC6 knock-down cells resulted in little to no rescue of aggresome formation. On the other hand, formation of ubiquitin-free ag- gresomes, which appear after expression of the aggregation-prone GFP chimera GFP-250, which is not ubituitylated (Garcia-Mata et al., 1999), does not seem to require HDAC6 at all (Kawaguchi et al., 2003). Then again, the inhibition of tubulin deacetylase activity has a profound effect on the transport of LC3, a component of the autophagy apparatus, to the aggresome, but affects the transport of polyubiquitylated misfolded huntingtin to a much lesser extent (Iwata et al., 2005). We therefore decided to investigate whether the catalytic activity 85
Chapter 3 of HDAC6 was required for its interaction with FAT10. In fact, this turned out not to be the case. A catalytically dead mutant, in which the deacetylase activity was inactivated by two point mutations in the active sites, interacted with FAT10 just as well as the catalytically active wild-type HDAC6 (Fig. 21A). Treatment of cells with the broad-spectrum deacetylase inhibitor trichostatin A even lead to an increased association of HDAC6 with FAT10, which could already be observed in the absence of proteasome inhibition (compare Fig. 21B lanes 1 and 2 vs. 5 and 6). To elucidate whether this increase was a direct effect of HDAC6 inhibition or a secondary consequence of inhibiting one or more of the many deacetylases also affected by TSA, we repeated the experiment using the HDAC6 specific tubulin deacetylase inhibitor tubacin (Haggarty et al., 2003). Treatment of cells with tubacin had hardly any effect on the association of FAT10 with HDAC6 although it markedly increased the level of acetylated α-tubulin (Fig. 21C). The addition of neither inhibitor affected HDAC6-FAT10 interaction in in vitro pull-down exper- iments (Fig. 28), suggesting that the inhibition of catalytic activity has no influ- ence on the binding capacity of HDAC6 for FAT10. We therefore conclude that the deacetylase activity of HDAC6 is dispensable for its interaction with FAT10. FAT10 is acetylated, but acetylation is not required for interaction with HDAC6 The findings that (1) HDAC6 interacts with FAT10 through one of its catalytic domains, that (2) treatment with trichostatin A slightly increases the interaction between the two proteins and that (3) two-dimensional gel electrophoresis of FAT10 reveals several closely migrating bands with a similar molecular weight but distinct isoelectric points (Raasi et al., 2001) collectively raise the question if FAT10 is acetylated and whether this might be a prerequisite for its interaction with HDAC6. To address this question, we investigated whether immunoprecip- itated FAT10 showed reactivity with an antibody against acetylated lysine. As can be seen in Fig. 29A, FAT10 can indeed be acetylated, although this is in all likelihood not a very dynamic process, as treatment of cells with trichostatin A for 6 hours did not change the level of FAT10 acetylation. In addition, FAT10 does not appear to be a substrate of HDAC6 either, since FAT10 exhibited similar levels of acetylation when expressed in the presence or absence of HDAC6 (Fig. 29B). The degree of FAT10 acetylation is estimated to be low as judged from 2D gels (Raasi et al., 2001) and since exposure times for the anti-acetylated lysine Western blots (Figs. 21E and 29) were at least 10-fold longer than for the anti-HA blots. In fact, the pool of FAT10 which interacted with HDAC6 after proteasome inhibition did not appear to be acetylated at all, as indicated by the complete absence of anti-acetyl-lysine reactivity of the FAT10 which co- 86
Page 1 and 2:
Role of the ubiquitin-like modifier
Page 3 and 4:
Acknowledgements . . . . . . . . .
Page 5 and 6:
Summary / Zusammenfassung English F
Page 7 and 8:
Summary In conclusion, these result
Page 9 and 10:
Summary nicht von der katalytischen
Page 11 and 12:
The Ubiquitin-Proteasome-System The
Page 13 and 14:
Introduction Figure 1: The 26S prot
Page 15 and 16:
Introduction are the major source o
Page 17 and 18:
Introduction majority of ubiquitin-
Page 19 and 20:
Introduction example contained in t
Page 21 and 22:
Introduction two modifiers apart fr
Page 23 and 24:
Introduction Figure 4: Comparison o
Page 25 and 26:
Introduction dependent apoptosis in
Page 27 and 28:
Introduction Interestingly, a recen
Page 29 and 30:
The Autophagy-Lysosome System Intro
Page 31 and 32:
Introduction Figure 5: Molecular ma
Page 33 and 34:
Introduction The transport of misfo
Page 35 and 36: Introduction where the proteasome i
Page 37 and 38: Chapter 1 The UBA domains of NUB1L
Page 39 and 40: Introduction Chapter 1 Ubiquitin, a
Page 41 and 42: Chapter 1 no correlation between th
Page 43 and 44: Chapter 1 covered proteins were sep
Page 45 and 46: Chapter 1 Figure 8: Accelerated deg
Page 47 and 48: Chapter 1 Treatment with IFN-γ and
Page 49 and 50: Chapter 1 ing SUMO-1-GFP or the C-t
Page 51 and 52: Chapter 1 Figure 12: Accelerated de
Page 53 and 54: Chapter 1 Figure 13: Co-immunopreci
Page 55 and 56: Chapter 1 domains of NUB1L is requi
Page 57 and 58: Chapter 1 Rad23 (Verma et al., 2004
Page 59 and 60: Chapter 1 FAT10-GFP expressing vect
Page 61 and 62: Chapter 2 Degradation of FAT10 by t
Page 63 and 64: Introduction Chapter 2 The proteaso
Page 65 and 66: Chapter 2 From our previous data it
Page 67 and 68: Chapter 2 Figure 14: Purified compo
Page 69 and 70: Chapter 2 evaluation of the NUB1L b
Page 71 and 72: Chapter 2 teins which contain intri
Page 73 and 74: Chapter 2 Combined with our studies
Page 75 and 76: Chapter 2 Recombinant expression of
Page 77 and 78: Chapter 2 et al., 2003), both at a
Page 79 and 80: Abstract Chapter 3 During misfolded
Page 81 and 82: Chapter 3 tion between the UPS and
Page 83 and 84: Chapter 3 Figure 18: HDAC6 interact
Page 85: Chapter 3 Figure 19: Both the BUZ d
Page 89 and 90: Chapter 3 precipitates with HDAC6 (
Page 91 and 92: 90 Chapter 3
Page 93 and 94: Chapter 3 Figure 23: The model conj
Page 95 and 96: Chapter 3 HDAC6 is required for the
Page 97 and 98: Chapter 3 Figure 26: Both ubiquitin
Page 99 and 100: Chapter 3 also revealed endogenous,
Page 101 and 102: Chapter 3 and/or its conjugates is
Page 103 and 104: Chapter 3 (Hipp et al., 2005), pEGF
Page 105 and 106: Chapter 4 FAT10-deficient mice disp
Page 107 and 108: Chapter 4 Once the infection is cle
Page 109 and 110: Chapter 4 As both wild-type as well
Page 111 and 112: Chapter 4 Peptides. The synthetic p
Page 113 and 114: Introduction Chapter 5 Antibodies a
Page 115 and 116: Chapter 5 binant human and mouse GS
Page 117 and 118: Chapter 5 Figure 34: The antibodies
Page 119 and 120: Chapter 5 Figure 36: The antibody s
Page 121 and 122: Discussion 30 years ago, ubiquitin
Page 123 and 124: Discussion monomeric ubiquitin as w
Page 125 and 126: Discussion Alternatively, FAT10 cou
Page 127 and 128: References D. T. Akey, X. Zhu, M. D
Page 129 and 130: References Y.-H. Chiu, Q. Sun, and
Page 131 and 132: References S. Gunawardena, L.-S. He
Page 133 and 134: References J. A. Johnston, M. E. Il
Page 135 and 136: References F. Lévy, N. Johnsson, T
Page 137 and 138:
References J.-H. Park, H.-S. Jong,
Page 139 and 140:
References P. Schreiner, X. Chen, K
Page 141 and 142:
References S. Vijay-Kumar, C. E. Bu
Page 143 and 144:
Abbreviations aa amino acid AAA ATP
Page 145 and 146:
Amino Acids Alanine Ala A Arginine
show all

Role of the ubiquitin-like modifier FAT10 in protein degradation and ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?